Olsen Helle Birk, Leuenberger-Fisher Melissa R, Kadima Webe, Borchardt Dan, Kaarsholm Niels C, Dunn Michael F
Research & Development, Novo Nordisk A/S, Bagsvaerd, Denmark.
Protein Sci. 2003 Sep;12(9):1902-13. doi: 10.1110/ps.03116403.
3-Nitro-4-hydroxybenzoate (3N4H) is a probe of the structure and dynamics of the metal-centered His B10 assembly sites of the insulin hexamer. Each His B10 site consists of a approximately 12 A-long cavity situated on the threefold symmetry axis. These sites play an important role in the storage and release of insulin in vivo. The allosteric behavior of the insulin hexamer is modulated by ligand binding to the His B10 zinc sites and to the phenolic pockets. Binding to these sites drives transitions among three allosteric states, designated T(6), T(3)R(3), and R(6). Although a wide variety of mono anions bind to the His B10 zinc sites of R(3), X-ray structures of ligands complexed to this site exist only for H(2)O, Cl(-), and SCN(-). This work combines one- and two-dimensional (1)H NMR and UV-Vis absorbance studies of the structure and dynamics of the 3N4H complex, which establish the following: (1). relative to the NMR time scale, 3N4H exchange between free and bound states is slow, while flipping among three equivalent orientations about the site threefold axis is fast; (2). binding of 3N4H perturbs resonances within the His B10 zinc site and generates NOEs between ligand resonances and the insulin C-alpha and side chain resonances of ValB2, AsnB3, LeuB6, and CysB7; and (3).3N4H exchange for other ligands is limited by a protein conformational transition. These results are consistent with coordination of the 3N4H carboxylate to the His B10 zinc ion and van der Waals interactions with Val B2, Asn B3, Leu B6, and Cys A7.
3-硝基-4-羟基苯甲酸酯（3N4H）是胰岛素六聚体中以金属为中心的His B10组装位点的结构和动力学探针。每个His B10位点由位于三重对称轴上的一个约12埃长的腔组成。这些位点在体内胰岛素的储存和释放中起重要作用。胰岛素六聚体的变构行为受配体与His B10锌位点和酚口袋的结合调节。与这些位点的结合驱动了三种变构状态之间的转变，分别称为T(6)、T(3)R(3)和R(6)。尽管多种单阴离子与R(3)的His B10锌位点结合，但仅存在与该位点络合的配体的H(2)O、Cl(-)和SCN(-)的X射线结构。这项工作结合了一维和二维（1）H NMR以及3N4H络合物结构和动力学的紫外可见吸收研究，得出以下结论：（1）相对于NMR时间尺度，3N4H在游离态和结合态之间的交换缓慢，而围绕位点三重轴在三个等效取向之间的翻转快速；（2）3N4H的结合扰乱了His B10锌位点内的共振，并在配体共振与ValB2、AsnB3、LeuB6和CysB7的胰岛素C-α和侧链共振之间产生了NOE；（3）其他配体的3N4H交换受蛋白质构象转变限制。这些结果与3N4H羧酸盐与His B10锌离子的配位以及与Val B2、Asn B3、Leu B6和Cys A7的范德华相互作用一致。
